P-197 Nuclear transfer with almost nonexistent mitochondrial carryover -a mouse model-

Abstract Study question Can the transfer of aggregated chromosomes (AC) as compared to spindle-chromosome (SCT) reduce mitochondrial DNA (mtDNA) carryover and improve development rates? Summary answer The rate mtDNA after AC was only 0.1%, which much less than that 2.0% SCT. What is known already Nuclear techniques such SCT pronuclear have been applied prevent diseases treat early embryonic arrest, however, problem has not yet resolved. We previously reported on an (ACT) method in humans could overcome above problem, consequent rates investigated Japan due legal restrictions no animal study conducted, formation ACs found any other species. design, size, duration Following our success creation mouse oocytes using IBMX (3-Isobutyl 1-methylxanthine), a phosphodiesterase inhibitor, model ACT established. This followed by comparison embryo both over 100 oocytes. distribution mitochondria around SC were also examined confocal laser microscope (FV3000, Olympus, Japan). Participants/materials, setting, methods GV stage collected from 8-12 week B6D2F1 mice. After GVBD, incubated KSOM medium, containing 3 mM IBMX, for 10-14 hours until formed. SCs injected into enucleated MII donor then fertilized at stage. Embryos cultured 5 days compare development. Enucleated subjected real-time PCR, analyze mtDNA. Main results role chance fertilization, cleavage blastocyst without (control), 60.7%, 55.9%, 13.2% 82.5%, 80.8% 30.8% (ACT), 81.0%, 74.5% 31.4% respectively. group showed significant increase fertilization (P = 0.007), (p 0.008) 0.034) rates, those control group. There differences between regarding formation. relative, PCR 0.1% ± 0.07%, significantly lower 0.6% 0.027). 3D microscopy revealed localized either or ACs, they pronuclei. Limitations, reasons caution Although we succeeded establishing ACT, validation human required confirm benefit this method. Further experiments are if healthy offspring can be obtained ACT. Wider implications findings amount heteroplasmy. As improved low caused had similar SCT, may give rise being employed novel nuclear transfer. Trial registration number applicatable